Markam, M. | Search bioRxiv

bioRxiv2025

Apoptosis reveals human evolutionary heritage sequestered in non-coding DNA

Cell-free chromatin particles (cfChPs), primarily derived from non-coding DNA (ncDNA) and released from apoptotic cells into the bloodstream, can be horizontally transferred into living cells. However, their potential role in generating ncDNA within recipient cells remains unknown. We previously reported that cfChP- sized fragments generated upon sonicating high-molecular-weight DNA can indiscriminately transmit themselves into foreign cells across species and kingdom boundaries. We hypothesized that cfChP-like DNA-protein complexes generated following organismal death have been exchanged among species and have progressively accumulated to form a dense patchwork that now constitutes the ncDNA, and that apoptosis may dissociate these complexes, rendering them amenable to detection. To test this hypothesis, we used species-specific DNA fluorescent in situ hybridisation probes and antibodies against archaea, eubacteria, plants, algae, fungi, protozoa, Drosophila, fish, chicken, mouse, rat, pig, dog, and monkey on intact and apoptotic human cells. We detected no reactivity with intact cells but strong reactivity with the ncDNA component of apoptotic cells. These findings indicate that ncDNA represents a dense conglomeration of DNA-protein complexes derived from different species that become detectable following apoptosis. This suggests that human evolutionary heritage is conserved within ncDNA formed through progressive accumulation of genetic fragments transferred horizontally following organismal death.

evolutionary biology↗

bioRxiv2025

Bendless-mediated K63 ubiquitination modulates cellular signalling to regulate Drosophila hematopoiesis

Ubiquitination is a reversible modification whose traditional role has been associated with K48-linked poly-ubiquitination involved in proteasomal degradation. However, the role of K63-linked poly-ubiquitination has been explored in various cellular processes like DNA repair, endocytosis, innate immune response, kinase activation, etc. Since K63-linked poly-ubiquitination can regulate the stability, localization, and activity of its target molecules, its regulation and function in various developmental and disease contexts are being explored. Here, we investigate how K63 ubiquitination regulates Drosophila blood cell homeostasis. Ubc13 (UBE2N), an E2 conjugating enzyme, is highly expressed in Acute Myeloid Leukemia (AML), wherein it controls innate immune signalling for the survival of AML cells; however, its role during developmental hematopoiesis is less explored. In Drosophila, Bendless is the functional homolog of Ubc13, whose role in stem cell regulation and particularly hematopoiesis is unknown. Our results indicate that spatial genetic perturbation of Bendless and its associated molecules, namely Uev1a, Effete, and E3 ligase - Traf6, that mediate K63 ubiquitination are critical for maintaining hematopoietic progenitors and regulating their differentiation. We show that Bendless-mediated K63 ubiquitination controls the Wingless signalling pathway by regulating Dishevelled in the larval lymph gland (LG) thereby modulating hematopoietic progenitor maintenance and differentiation. Furthermore, an excess of K63 ubiquitination activates the JNK pathway in the LG, resulting in lamellocyte production. Genetic epistasis analysis shows that activation of the canonical Wingless pathway in the background of Bendless depletion or inactivation of the JNK pathway in Bendless over-expression conditions can restore physiological hematopoiesis. Our findings indicate that regulators of K63 ubiquitination, like Bendless, could act as molecular inter-nodes that are capable of signalling cross-regulation, especially where intricate signalling networks are involved. Our study provides important mechanistic insights into the signalling mechanisms regulated by K63 ubiquitination during stem cell homeostasis.

developmental biology↗